Methods and compositions for short stature plants through manipulation of gibberellin metabolism to increase harvestable yield

Patent metadata

Field	Value
Publication number	US-11414670-B2
Application number	US-202016847283-A
Country	US
Kind code	B2
Filing date	Apr 13, 2020
Priority date	Aug 17, 2016
Publication date	Aug 16, 2022
Grant date	Aug 16, 2022

Abstract

The present disclosure provides compositions and methods for altering gibberellin (GA) content in corn or other cereal plants. Methods and compositions are also provided for altering the expression of genes related to gibberellin biosynthesis through suppression, mutagenesis and/or editing of specific subtypes of GA20 or GA3 oxidase genes. Modified plant cells and plants having a suppression element or mutation reducing the expression or activity of a GA oxidase gene are further provided comprising reduced gibberellin levels and improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types.

First claim

What is claimed is:

1. A method for producing a transgenic corn plant, comprising:
(a) transforming at least one cell of a corn explant with a recombinant DNA construct comprising a transcribable DNA sequence encoding a non-coding RNA molecule, wherein the non-coding RNA molecule comprises a targeting sequence that is:
(i) at least 80% complementary to at least 19 consecutive nucleotides of a first mRNA molecule encoding a first endogenous gibberellin (GA) oxidase protein in a corn plant, the first endogenous GA oxidase protein being at least 90% identical to SEQ ID NO: 9; and
(ii) at least 80% complementary to at least 19 consecutive nucleotides of a second mRNA molecule encoding a second endogenous GA oxidase protein in a corn plant, the second endogenous GA oxidase protein being at least 90% identical to SEQ ID NO: 15;

wherein the transcribable DNA sequence is operably linked to a plant-expressible promoter; and
(b) regenerating or developing the transgenic corn plant from the transformed explant, wherein the transgenic corn plant comprises the recombinant DNA construct.

2. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 19 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 13, and 14.

3. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 19 consecutive nucleotides of the first mRNA molecule encoding the first endogenous GA20 oxidase protein.

4. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is 100% complementary to at least 19 consecutive nucleotides of the first mRNA molecule encoding the first endogenous GA20 oxidase protein.

5. The method of claim 3 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 19 consecutive nucleotides of SEQ ID NO: 7 or 8.

6. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 19 consecutive nucleotides of the second mRNA molecule encoding the second endogenous GA20 oxidase protein.

7. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is 100% complementary to at least 19 consecutive nucleotides of the second mRNA molecule encoding the second endogenous GA20 oxidase protein.

8. The method of claim 6 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 19 consecutive nucleotides of SEQ ID NO: 13 or 14.

9. The method of claim 1 , wherein the non-coding RNA molecule encoded by the transcribable DNA sequence is a precursor miRNA or siRNA that is processed or cleaved in a plant cell to form a mature miRNA or siRNA.

10. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is at least 90% complementary to at least 21 consecutive nucleotides of the first mRNA molecule encoding the first endogenous GA20 oxidase protein, and is at least 90% complementary to at least 21 consecutive nucleotides of the second mRNA molecule encoding the second GA20 oxidase protein.

11. The method of claim 1 , wherein the targeting sequence of the non-coding RNA molecule is 100% complementary to at least 21 consecutive nucleotides of the first mRNA molecule encoding the first endogenous GA20 oxidase protein and is 100% complementary to at least 21 consecutive nucleotides of the second mRNA molecule encoding the second GA20 oxidase protein.

12. The method of claim 1 , wherein the plant-expressible promoter comprises a vascular promoter.

13. The method of claim 12 , wherein the vascular promoter is a rice tungro bacilliform virus (RTBV) promoter.

14. The method of claim 12 , wherein the vascular promoter is selected from the group consisting of: a sucrose synthase promoter, a sucrose transporter promoter, a Sh1 promoter, Commelina yellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV) large intergenic region (LIR) promoter, a maize streak geminivirus (MSV) coat protein (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter, and a rice yellow stripe 2 (OsYSL2) promoter.

15. The method of claim 12 , wherein the vascular promoter comprises a DNA sequence that is at least 80% identical to SEQ ID NO: 65 or SEQ ID NO: 66.

16. The method of claim 12 , wherein the vascular promoter comprises a DNA sequence that is at least 90% identical to SEQ ID NO: 65 or SEQ ID NO: 66.

17. The method of claim 12 , wherein the vascular promoter comprises a DNA sequence that is at least 80% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71.

18. The method of claim 12 , wherein the vascular promoter comprises a DNA sequence that is at least 90% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71.

19. The method of claim 1 , wherein the plant-expressible promoter comprises a constitutive promoter.

20. The method of claim 19 , wherein the constitutive promoter comprises a promoter selected from the group consisting of: an actin promoter, a CaMV 35S or 19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a FMV promoter, a CMV promoter, a MMV promoter, a PCLSV promoter, an Emu promoter, a tubulin promoter, a nopaline synthase promoter, an octopine synthase promoter, a mannopine synthase promoter, and a maize alcohol dehydrogenase, or a functional portion thereof.

21. The method of claim 19 , wherein the constitutive promoter comprises a nucleotide sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83.

22. The method of claim 1 , wherein the plant-expressible promoter comprises a leaf promoter.

23. The method of claim 22 , wherein the leaf promoter is selected from the group consisting of: a RuBisCO promoter, a PPDK promoter, a FDA promoter, a Nadh-Gogat promoter, a chlorophyll a/b binding protein gene promoter, a phosphoenolpyruvate carboxylase (PEPC) promoter, or a Myb gene promoter.

24. The method of claim 22 , wherein the leaf promoter comprises a nucleotide sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74.

25. The method of claim 1 , wherein the at least one cell of the explant is transformed via Agrobacterium mediated transformation or Rhizobium -mediated transformation.

26. The method of claim 1 , wherein the at least one cell of the explant is transformed via microprojectile-mediated transformation or particle bombardment-mediated transformation.

27. The method of claim 1 , wherein the non-coding RNA molecule reduces the expression levels of the first and second mRNA molecules in at least one tissue of the transgenic corn plant, relative to a control plant, when the non-coding RNA molecule is expressed in the transgenic corn plant.

28. The method of claim 1 , wherein expression of the non-coding RNA molecule in the corn plant reduces the level of one or more active GAs in the transgenic corn plant, relative to a control plant, when the non-coding RNA molecule is expressed in the transgenic corn plant.

29. The method of

Assignees

• Monsanto Technology Llc

Inventors

• Allen Edwards M
• Boddu Jayanand
• Dietrich Charles R
• Goldshmidt Alexander
• Howell Miya
• Kosola Kevin R
• Neelam Anil
• Slewinski Thomas L
• Venkatesh Tyamagondlu V
• Wang Huai
• Manjunath Sivalinganna
• Rymarquis Linda

Classifications

• C12N15/1137

  against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138) · CPC title

• C12N15/8261 Primary

  with agronomic (input) traits, e.g. crop yield · CPC title

• C12N15/8273 Primary

  for drought, cold, salt resistance · CPC title

• C12N15/8297 Primary

  Gibberellins; GA3 · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title